The present invention relates to a liquid composition for intravenous administration which comprises an aqueous solution of immunoglobulin. The immunoglobulin is generally immunoglobulin G (IgG) derived from human blood plasma.
Immunoglobulin for intravenous infusion has been in clinical use for several years. The product is available as a lyophilised dry formulation or in some cases, as an intravenously injectable liquid formulation. The dry formulation, presented as a vial containing for example 5 g of immunoglobulin, requires to be reconstituted into an injectable solution before use and doses of up to 1 g per Kg body weight per day are recommended for several clinical indications. Such large doses require a number of vials to be reconstituted into an injectable formulation, which is inconvenient and time consuming. There are therefore considerable advantages in providing a ready to use injectable formulation. However, such liquid formulations require to be stable on long term storage.
Conventionally, IgG is isolated from blood plasma using ethanol fractionation processes. These include the original Cohn-Oncley methods which are still in use principally in the USA and various other established modifications of that method which are used mainly in Europe (for a review of ethanol fractionation processes see Ethanol Precipitation by Kistler P. and Friedli H. in Methods of Plasma Protein Fractionation, J. M. Curling, ed., Academic Press, Inc., New York, 1980). A disadvantage of ethanol, namely its potential for denaturing proteins, is offset by using low processing temperatures and hence these methods are generally referred to as xe2x80x9ccold ethanol fractionationxe2x80x9d. Cold ethanol fractionation processes all depend on the manipulation of five variables, ie. ethanol concentration, pH, ionic strength, temperature and protein concentration to achieve the selective separation of proteins into different precipitates which, by convention, are known as Fractions. Fraction II is the principal immunoglobulin bearing precipitate in the cold ethanol fractionation of human blood plasma.
For some time preparations for the intramuscular administration of immunoglobulin have been formulated from Fraction II. However, the intravenous infusion of these preparations has been found to cause severe adverse reactions, resembling anaphylaxis (ie. cardiovascular collapse and bronchospasm), in recipients (see Immunoglobulins: Characteristics and uses of intravenous preparations, Alving B. M. and Finlayson J. S., eds, US Dept. of Health and Human Sciences Publication No. (FDA)-80-9005, 1979). These severe adverse reactions are now known to be caused principally by the presence of aggregates of IgG molecules and the contamination of Fraction II with trace quantities of vasoactive blood plasma enzymes such as pre-kallikrein activator (PKA) and kallikrein. Aggregated immunoglobulin can bind and activate the complement group of blood plasma proteins (so called xe2x80x9canti-complementary activityxe2x80x9d) and the activation of the complement system results in the generation of the complement peptides C5a and C3a which are anaphylatoxins. It is also known that the administration of PKA and kallikrein in physiological significant quantities can cause severe hypertension and cardiovascular collapse.
Thus in the preparation of a formulation of immunoglobulin for intravenous infusion attention needs to be paid to the above issues. A number of approaches have been taken to solving this problem. These include, altering the processing of Fraction II in order to prevent aggregate formation; further purification of the immunoglobulin from Fraction II so as to remove aggregates and other contaminating plasma proteins and the treatment of immunoglobulin from Fraction II with very low levels of a proteolytic enzyme such as pepsin to dissociate any aggregates and residual PKA and kallikrein. (For a review of the production of Immunoglobulin for intravenous infusion see Methods for the Production of IVIG Preparations and Analysis of IVIG Preparations Available, by Lundblad J. L. and Schroeder D. D. in Clinical applications of intravenous immunoglobulin therapy, P. L. Yap, ed., Churchill Livingstone Inc., New York, 1992). The use of pepsin in this way has been found to be optimum at a relatively low pH, eg. 4.0. Additionally it is well understood in the art that such a low pH treatment is an effective virus inactivation procedure (see Reid, K. G. et al. Vox Sang. 55 p75-80 , 1988. Potential contribution of mild pepsin treatment at pH4 to the viral safety of human immunoglobulin products).
In fact, preparations of human immunoglobulin for intravenous infusion are required to meet certain standards, such as those recommended by the European Pharmacopoeia Commission which sets out guidelines for inter alia distribution of molecular size, anti-complementary activity, PKA and that the method of preparation includes a step or steps that have been shown to inactivate known agents or infection (see European Pharmacopoeia Third Edition published June 1996 to replace the second edition on Jan. 1, 1997, Monograph number 1997: 0918, Human Normal Immunoglobulin for Intravenous Administration).
The majority of human immunoglobulin products for intravenous infusion on the market currently are in the form of freeze dried preparations to provide stability on shipment and storage. These preparations must be reconstituted before use which can be inconvenient and time consuming as described earlier. In addition, liquid compositions of immunoglobulin for intravenous infusion are also available.
U.S. Pat. No. 4,499,073 (Cutter Laboratories Inc.) describes the production of an intravenous injectable solution of human immunoglobulin which is required to have a pH in the range 3.5 to 5.0. Furthermore, the ionic strength is required to be reduced to low levels, particularly below 0.001. The maintenance of the pH within this range and low ionic strength are said to be essential to the ability to store the liquid.
Formulation for extended periods, whilst satisfying criteria such as distribution of molecular size and anti-complementary activity.
Another proposal for the production of a stable liquid formulation of human immunoglobulin is contained in W095/22990 (The Green Cross Corporation) which requires a pH in the region 5.5 in conjunction with a low electrical conductivity of less than 1 mmho.
The proposals set out in U.S. Pat. No. 4,499,073 relate to the treatment of Fraction II or Fraction III filtrate (Supernatant III) produced using the methods described originally by E. J. Cohn et al (J. Am. Chem. Soc. 68 :459-475, 1946) and, L. J. Oncley et al (J. Am. Chem. Soc. 71: 541-550, 1949). However, these conditions do not appear to be suitable for the production of a stable IgG solution derived from other cold-ethanol fractionation schemes. The pH conditions and low ionic strength specified in this reference do not result in the formation of a stable product when applied to immunoglobulin prepared according to the cold ethanol fractionation scheme used by the present applicants. Since different modified cold ethanol fractionation methods are used widely, especially in Europe, there is therefore a need for a stable IgG solution derived from starting materials other than those taught as suitable in the prior art.
It has now been surprisingly discovered that stable intravenously injectable immunoglobulin solutions may be obtained by employing quite different conditions of pH and ionic strength to those taught in the prior art, with the additional inclusion of treating the immunoglobulin preparation with an enzyme such as pepsin.
Thus, the present invention provides a liquid composition for intravenous administration which comprises a solution of an immunoglobulin in a pharmaceutically acceptable aqueous carrier, the solution having a pH in the range 5.0 to 5.8 and an ionic strength On the range 0.02 to 0.25, the immunoglobulin having been subjected to treatment with pepsin.
The ionic strength may be in the range 0.04-0.25. Ionic strength (I) is defined as half the sum of the terms obtained multiplying the concentration of ion (C) in a solution by the square of its valency (Z) ie. I={fraction (1/2 )}xcexa3C. Zxe2x88x92. For example, the ionic strength of 60mM NaCl would be calculated as follows: xc2xd[0.06xc3x971xe2x88x92)+(0.06xc3x971xe2x88x92)]=0.06
Solutions of immunoglobulin prepared according to the present invention have been measured as having conductivity values of approximately 4 mmho to over 20 mmho.
The aqueous carrier must be pharmaceutically acceptable in the sense of being compatible with other ingredients of the composition and not injurious to the patient.
The liquid composition of the present invention has the advantage of not being freeze dried. Thus, the liquid composition does not have to be reconstituted prior to use. Chemical modification of the immunoglobulin is not required nor is extensive additional purification of the Fraction II.
It has advantageously beer. found that formulation of the liquid composition according to the present invention results in a composition which is stable upon storage. Stable upon storage is taken to mean that the immunoglobulin does not substantially aggregate nor degrade and maintains acceptable levels of anti-complementary activity, PKA activity and kallikrein activity during storage for an extended period at a temperature in the range 4xc2x0 C. to 25xc2x0 C.
An extended period is taken to mean 12 weeks, preferably 26 weeks and most preferably 52 weeks at 25xc2x0 C.; also 6 months, preferably 12 months and most preferably 24 months at 4xc2x0 C.
That the composition does not substantially aggregate is taken to mean that there is an increase of no more than 2.5% to 3% in the immunoglobulin content of the preparation which has a molecular size greater than the IgG dimers present in the preparation. That the composition does not substantially degrade is taken to mean that no more than 5% to 7% of the preparation has a molecular size less than the IgG monomers present in the preparation. In addition the composition is considered to have shown acceptable levels of degradation if more than 50% of the initial antibody function (eg. anti-rubella virus activity) remains. An acceptable level of anti-complementary activity is one where the consumption of complement is not greater than 50% (1 CH50 per milligram of immunoglobulin: for definition see protocol B attached hereto). Acceptable levels of PKA and kallikrein are not more than 35 iu per mL and less than 0.05 iu per mL respectively in a solution containing 30 g/L of immunoglobulin. Tests for distribution of molecular size, anti-complementary activity, PKA/kallikrein activity and anti rubella activity are described in the protocols A to D below.
A liquid composition which showed the stability characteristics described above would have the potential to meet certain standards (such as those referred to previously in the European Pharmacopoeia) for formulations of immunoglobulin suitable or intravenous infusion.
In accordance with the above requirements regarding acceptable levels of aggregation, anti-complementary activity, PKA/kallikrein content and antibody function, preferably the pH of the liquid composition is in the range pH 5.25-5.75 and the ionic strength is ;n the range 0.04 -0.18, more preferably 0.03-0.18.
It has now been found that pepsin treatment together with formulation of the composition as defined herein obviates any requirement to employ further purification or modification to the Fraction II material. The liquid formulation may contain residual pepsin but this can be removed if necessary.
Additionally, treatment with pepsin at low pH, eg. pH4 is particularly effective as a virus inactivation step.
The immunoglobulin of the composition is preferably IgG which may contain residual amounts of other immunoglobulins eq. IgA and/or IgM up to a level of 5% by weight of the total immunoglobulin content. However, compositions suitable for a given particular use may comprise IgA and/or IgM as required.
The immunoglobulin of the liquid composition may be obtained by way of any suitable method. For example, the immunoglobulin may be prepared according to the Cohn-Oncley cold ethanol fractionation process mentioned previously or a modified method of cold ethanol fractionation such as that described in FIG. 1. The immunoglobulin may be obtained as a Fraction II precipitate or as a supernatant (for example Supernatant III or I and III) from the fractionation process.
The Fraction II precipitate may be frozen and stored prior to resolution, removal of residual ethanol impurity, treatment with pepsin and formulation into the liquid composition suitable for intravenous administration. The ethanol impurity in the redissolved Fraction II (or supernatant I and III) may be removed, for example, by diafiltration of a solution thereof against a suitable buffer at a low pH. A suitable buffer contains for example 0.45% NaCl, 1% sucrose, and 200-400 ml 1M HCl/kg protein. Alternatively, for example, the ethanol impurity may be removed by freeze drying and the dried solids redissolved and adjusted to a low pH using a suitable titrant such as hydrochloric acid. Generally speaking the pH of the immunoglobulin solution from which the ethanol impurity has been removed is in the range 3.9 to 4.5 and preferably 3.9 to 4.1 and the residual ethanol impurity less than 10 mg/g of protein.
The solution comprising immunoglobulin may then be concentrated (eg. by ultrafiltration) to 8-12% w/w total protein and a suitable stabiliser such as a carbohydrate (eg. glucose, maltose or sucrose) added at a concentration of between one part stabiliser per one part protein (w/w) to two parts stabiliser per part protein (w/w). For example 1:1 (w/w) glucose to protein, 1.5:1 or 2:1 maltose to protein and 1.5:1 or 2:1 sucrose to protein. Other stabilisers such as amino acids (eg. glycine) may also be added at this stage at a concentration of up to 100 mg/g protein.
Incubation with a small amount of pepsin is then carried out to reduce the formation of aggregates of immunoglobulin monomer; reduce anticomplementary activity and decrease levels of PKA and kallikrein activity. Preferably pepsin is added in the range 10-150 xcexcg/g total protein and more preferably in the range 25-100 xcexcg/g total protein. The immunoglobulin solution is then incubated at a suitable temperature in the range 20xc2x0 C. to 37xc2x0 C. and preferably 35xc2x0 C. and for a suitable time in the range 1 hour to 72 hours and preferably 20-24 hours.
After incubation the pH. of the immunoglobulin solution is adjusted to between pH 5.0 to 5.8 using a suitable titrant, for example, sodium hydroxide. Preferably the pH is adjusted to between pH 5.25-5.75. The protein concentration of the solution may then be adjusted to 2-10% (w/w) and preferably to 4-6% (w/w) by dilution using a salt solution of appropriate concentration so as to bring the ionic strength of the solution to between 0.02 and 0.25. Preferably the ionic strength is adjusted to between 0.03 and 0.18. If other stabilisers such as amino acids (eg. glycine) have not been added to the composition earlier (see above) they can be included in the formulation at this stage at a concentration of up to 100 mg/g protein.
The formulated immunoglobulin solution is then filtered to remove any potential bacterial contamination and aseptically dispensed into pharmaceutically acceptable containers.
There is provided therefore a liquid composition for intravenous administration which is stable on storage and which comprises a solution in a pharmaceutically acceptable aqueous carrier of an immunoglobulin prepared from cold ethanol fractionation of plasma and which immunoglobulin does not require to be freezed dried in the final container